Cell surface layer-localized proteins are proteins that are present and immobilized in a cell surface layer. An example thereof is α- or a-agglutinin, which is a flocculation protein of yeast. Such proteins are similar to secretory proteins in that they have secretion signal sequences, but are different from secretory proteins in that they are transported while being immobilized in a cell membrane via a GPI anchor. In general, cell surface layer-localized proteins have a GPI anchoring domain on the C-terminal portion. Cell surface layer-localized proteins are immobilized on a cell membrane in the following manner: when the protein to be localized on the cell surface layer is passing across a cell membrane, a part (i.e., a GPI anchor attachment recognition signal sequence) of the domain of the protein is selectively cleaved, a newly projected C-terminal portion of the protein is bound to the GPI anchor on the cell membrane, and thus the protein is immobilized on the cell membrane. Then, the base portion of the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC). Then, the protein cleaved from the cell membrane is incorporated into the cell wall so as to be immobilized on the cell surface layer and thus is localized on the cell surface layer. Herein, “GPI anchor” refers to a glycolipid having ethanolamine phosphate−6 mannose α 1-2 mannose α 1-6 mannose α 14 glucosamine α 1-6 inositol phospholipid, which is also known as glycosylphosphatidylinositol (GPI), as the basic structure.
The GPI anchoring domain is generally positioned at or near the C-terminus of the cell surface layer-localized protein. For example, in addition to the GPI anchor attachment recognition signal sequence, there are four sugar chain-binding sites in the sequence encoding 320 amino acid residues from the C-terminus of α-agglutinin. These sugar chain-binding sites and polysaccharides constituting the cell wall are covalently bonded after the GPI anchor is cleaved by PI-PLC, so that the C-terminal sequence portion of α-agglutinin is bonded to the cell wall and thus the α-agglutinin is retained on the cell surface layer.
The inventors succeeded in expressing lipase on the cell surface layer, utilizing such a GPI anchoring domain (Japanese Laid-Open Patent Publication No. 11-290078). More specifically, the structural gene of lipase was placed upstream of the DNA encoding the GPI anchoring domain, and a secretion signal sequence was placed further upstream, so that the lipase was expressed on the cell surface layer such that the N-terminus thereof was outside the cell.